Immunization of fish with plant-expressed recombinant proteins

ABSTRACT

Plants are produced that express an amino acid sequence that, when administered to a fish, produce an antigenic or immune response in the fish. The amino acid sequence in one embodiment is an antigen from an organism that causes pathology in fish. The plant tissue may be fed to the fish, or mixed with other materials and fed to fish, or extracted and administered to the fish.

This application is a divisional application of previously filed andcopending application U.S. Ser. No. 10/733,031, which claims priority toU.S. Patent Application 60/433,381 filed on Dec. 13, 2002 both of whichare incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to the expression of fish disease antigens intransgenic plants and the use of the same as a vaccine.

BACKGROUND OF THE INVENTION

Over the past decade, transgenic plants have been successfully used toexpress a variety of useful proteins. For example, production ofproteases in plants has been achieved (See U.S. Pat. No. 6,087,558);along with production of aprotinin in plants (U.S. Pat. No. 5,824,870);and avidin (U.S. Pat. No. 5,767,379). A variety of mammalian bacterialand viral pathogen antigens are included in those proteins that havebeen successfully produced in plants, such as viral vaccines (U.S. Pat.No. 6,136,320), transmissible gastroenteritis and hepatitis vaccines(U.S. Pat. Nos. 5,914,123 and 6,034,298). These patents, as well as allreferences cited herein are incorporated herein by reference.

Many of the resulting peptides induced an immunogenic response in mice(Mason et al. (1998) Vaccine 16:13361343; Wigdorovitz et al. (1999)Virology 155:347-353), and humans (Kapusta et al. (1999) FASEB J.13:1796-1799) comparable to that of the original pathogen. After oraldelivery, these edible vaccines were immunogenic and could induceprotection. Mice fed a basic diet plus corn expressing recombinantEscherichia coli heat-labile enterotoxin B-subunit (LtB) mounted a gooddose dependent IgG and IgA response (Streatfield et al. “Plant basedvaccines—unique advances” Vaccine (2001)19:2742-2748.) Some of the firstedible vaccine technologies developed include transgenic potatoesexpressing hepatitis, TGEV and Norwalk virus antigens as well as variousother viral antigens. (See, e.g., Thanavala et al. (1995) Proc. Natl.Acad. Sci. U.S.A. 92:3358-3361; U.S. Pat. No. 6,136,320; U.S. Pat. No.6,034,298; U.S. Pat. No. 5,914,123; U.S. Pat. No. 5,612,487 and U.S.Pat. No. 5,484,719; Mason et al., (1996) Proc. Natl. Acad. Sci.93:5335-5340; “VP1 protein for foot-and-mouth disease” (Wigdorovitz etal (1999) Virology 255:347-353).

The utilization of transgenic plants for vaccine production has severalpotential benefits over traditional vaccine production methods. First,transgenic plants are usually constructed to express only a smallantigenic portion of the pathogen or toxin, eliminating the possibilityof infection or innate toxicity of the whole organism and reducing thepotential for adverse reactions. Second, since there are no known humanor animal pathogens that are able to infect plants, concerns with viralor prion contamination are eliminated. Third, immunogen production intransgenic crops relies on the same established technologies to sow,harvest, store, transport, and process the plant as those commonly usedfor food crops, making transgenic plants a very economical means oflarge-scale vaccine production. Fourth, expression of immunogens in thenatural protein-storage compartments of plants maximizes stability,minimizes the need for refrigeration and keeps transportation andstorage costs low. Fifth, formulation of multicomponent vaccines ispossible by blending the seed of multiple transgenic plant lines into asingle vaccine. Sixth, direct oral administration is possible whenimmunogens are expressed in commonly consumed food plants, such asgrain, leading to the production of edible vaccines.

Oral vaccine delivery as the primary or booster immunization is by farthe most sought after method by the aquaculture industry because it issuitable for the mass immunization of fish of all sizes, it is lessstressful on fish than injection delivery, which requires handling ofthe fish, and because it induces mucosal immunity. However thecost-effectiveness of oral delivery has been a major barrier tocommercialization of this method, especially for larger fish. Efficacyof oral antigen delivery is reported to be limited by the destructionand absorption of the antigens by the fish digestive system.

The inventors have found that transgenic plants can provide an idealsystem for economical production of antigens for oral vaccination offish.

BRIEF DESCRIPTION OF THE INVENTION

According to one aspect of the invention there is provided use of aplant-derived recombinant amino acid sequence in the manufacture of amedicament for the prevention or treatment of disease in fish, whereinthe amino acid sequence, when administered to fish, produces anantigenic or immunogenic response in the fish. Preferably therecombinant amino acid sequence is an antigen of an organism that causesdisease or pathology in fish.

In one aspect of the invention a plant is transformed with a nucleotidesequence encoding an amino acid sequence which, when administered to afish, produces an antigenic or immunogenic response in the fish.

In a further aspect of the invention, expression of the amino acidsequence is preferentially directed to the seed of the plant.

In another aspect, the invention provides an amino acid sequence derivedby expression in a plant cell, wherein said amino acid sequence isendogenous to an organism causing disease or pathology in fish.

In another aspect, the invention provides a composition suitable fororal delivery to fish, comprising a plant-derived recombinant amino acidsequence, in particular a plant-derived recombinant amino acid sequencewhich is an antigen of an organism that causes disease or pathology in afish.

In yet another aspect, the invention provides a method of immunizingfish against disease, which comprises administering to a fish acomposition comprising a plant-derived recombinant amino acid sequencewhich is an antigen of an organism that causes disease or pathology in afish.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the barley alpha amylase sequence fused to a sequenceencoding the avidin mature protein (SEQ ID NO: 1).

FIG. 2 is a plasmid map of pPHI5158.

FIG. 3 shows the maize optimized pat sequence (SEQ ID NO: 2).

FIG. 4 is a plasmid map of PGN7101.

FIG. 5A is the nucleotide sequence of maize codon optimized LtB (SEQ IDNO: 3).

FIG. 5B is the nucleotide sequence of BAASS:LtB (SEQ ID NO: 4).

FIG. 6 is the nucleotide sequence of IPNV VP2 (SEQ ID NO: 5).

FIG. 7 is the nucleotide sequence of BAASS:VP2 (SEQ ID NO: 6).

FIG. 8 is the nucleotide sequence of IPNV VP3 (SEQ ID NO: 7).

FIG. 9 is the nucleotide sequence of BAASS:VP3 (SEQ ID NO: 8).

FIG. 10 is the plasmid map of PGN9084.

FIG. 11 is the plasmid map of PGN9111.

FIG. 12 is a Western blot of the VP2 and VP3 proteins expressed in seed,resulting from event NVA.

FIG. 13 is a Western blot of the VP2 and VP3 proteins expressed in seed,resulting from event NVB.

FIG. 14 is a graph showing mean weight of fish at the time 0 (first bar)and 8 weeks after vaccination (second bar). Standard error bars are alsoshown

FIG. 15 are graphs showing mean antibody response of Atlantic salmon at8 weeks post-injection or feeding of recombinant avidin (A) or LtB (B)expressed in corn as measured by ELISA. Bars represent the standarderror of the mean. The number of animals sampled in each group (N) isindicated for each group.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

By use of the term “fish” herein is meant fin-fish, shellfish, and otheraquatic animals. Fin-fish include all vertebrate fish, which may be bonyor cartilaginous fish. The prime candidate fin-fish species forreceiving the vaccine of the invention are salmonid fish, includingsalmon and trout species, particularly coho salmon (Oncorhynchuskisutch), brook trout (Salvelinus fontinalis), brown trout (Salmotrutta), chinook salmon (Oncorhynchus tshawytscha), masu salmon(Oncorhyncus masou), pink salmon (Oncorhynchus gorbuscha), rainbow trout(Oncorhynchus mykiss), Arctic charr (Salvelinus alpinus) and Atlanticsalmon (Salmo salar). However, any other fish species susceptible toinfectious disease may benefit, such as ornamental fish species, koi,goldfish, carp, catfish, yellowtail, sea bream, sea bass, pike, halibut,haddock, tilapia, turbot, wolffish, and so on.

Examples of shellfish include, but are not limited to clams, lobster,shrimp, crab and oysters. Other cultured aquatic animals include, butare not limited to eels, squid and octopi.

A “plant-derived” recombinant amino acid sequence is an amino acidsequence engineered to be expressed in a transgenic plant whose sequenceis not endogenous to the plant.

An amino acid sequence of the invention is one which, when administeredto a fish, results in an antigenic or immunogenic response in the fish.

Antigens of organisms causing pathologies in fish and nucleotidesequences encoding such antigens have been administered to fish thathave been exposed by way of injection, immersion, spray, adding thevaccine directly to fish food, or gene transfer into fish cells. Forexample, U.S. Pat. No. 6,462,027 describes a method of contacting anisolated non-infectious polynucleotide encoding an immunogen with anaquatic animal. U.S. Pat. No. 6,180,614 describes introducing DNAplasmids encoding antigen-based vaccines by transfection into the fish.The promoter is one capable of directing expression in the fish. Thepatent specification notes that bacterially-expressed recombinantproteins can form inclusion bodies so that recovery of protein is low ornonexistent. Further, it indicates induction of an immune response mayrequire that the antigenic protein be correctly glycosylated and folded,which, they state, may not be accomplished in a cell other than ananimal cell. However, the inventors here have found that it is possibleto produce in a plant a correctly processed antigenic amino acidsequence that can cause an antigenic or immunogenic response whenadministered to fish.

The coding sequences of many amino acid sequences producing an antigenicor immunogenic response in fish (also referred to as an “antigen”) havebeen and are being sequenced, as there has been a great interest inproducing vaccines using such genes. While specific examples are setforth below to illustrate the principle of the invention using certainantigens, the invention is not limited to any particular antigen. Ratherany amino acid sequence that produces an antigenic or immune response ina fish can be used. In a preferred embodiment, an antigen of an organismcausing pathologies in fish is used. Such an antigen is used to induceor enhance immunity, and the corresponding nucleotide sequence whichencodes that antigen is useful in the invention. A few of the numerousexample of such sequences which have been isolated include the cDNAencoding structural protein-1 of infectious salmon anemia virus (ISAV)described in U.S. Pat. No. 6,471,964, as well as those discussed inTucker et al. (2000) “Assessment of DNA vaccine potential for juvenileJapanese flounder Paralichthys olivaceus, through the introduction ofreporter genes by particle bombardment and histopathology” Vaccine19(7-8):801-809; Corbeil et al. (1999) “Evaluation of the protectiveimmunogenicity of the N, P, M, NV, G proteins of infectioushematopoietic necrosis virus in rainbow trout Oncorhynchus mykiss usingDNA vaccines” Dis. Aquat. Organ 39(1):29-26; Nusbaum et al. (2002)“Protective immunity induced by DNA vaccination of channel catfish withearly and late transcripts of the channel catfish herpes virus (IHV-1)”Vet Immunol. Immunopathol 84(3-4):151-168; Clark et al. (1992)“Developmental expression of surface antigen genes in the parasiticcilate Ichtyophthirius multifiliis” Proc. Natl. Acad. Sci.89(14):6363-6367; and Sato et al. (2000) “Expression of YAV proteins andvaccination against viral ascites among cultured juvenile yellowtail”Biosci. Biotechnol. Biochem. 64(7):1494-1497.

Examples of the variety of pathogens for which the methods of theinvention can be useful include, without limitation, hemorrhagicsepticemia virus (VHSV), infectious pancreatic necrosis virus (IPNV),infectious haematopoietic necrosis virus (IHNV), salmon pancreas diseasevirus (SPDV), virus causing spring viremia of carp, grass carphemorrhagic virus, nodaviridae such as nervous necrosis virus or stripedjack nervous necrosis virus, infectious salmon anaemia virus (ISAV),Aeromonis salmonicida, Renibacterium salmoninarum, Yersinia spp.,Pasteurella spp. (including Photobacterium damselae), Vibrio spp.(including V. anguillarum and V. ordalii), Edwardsiella spp. (includingE. ictaluri and E. tarda), Piscirickettsia salmonis (causative ofSalmonid Rickettsial Septicaemia), Iridovirus, cardiomyopathy syndromevirus, taura syndrome virus, Penaeus monodon virus, shrimp yellowheadvirus, shrimp whitespot virus, and Streptococci spp.

Other examples of known antigens that produce pathology in fish that canbe used in the invention include: IPNV VP2 and VP3 proteins, IHNV Gprotein, VHSV G protein, Nodavirus capsid protein, ISAV antigensdisclosed in WO 01/10469, SPDV antigens disclosed in WO 99/58639, P.salmonis antigens disclosed in WO 01/68865, and Whitespot Virus antigensdisclosed in WO 01/09340. Numerous nucleic acid and amino acid sequencesof fish pathogen antigens are known and accessible through the Genbankdatabases and other sources.

An amino acid sequence or antigen of the invention which is “of anorganism causing disease or pathology in fish” is an amino acid sequenceor antigen of a pathogen of fish (or a derivative thereof), which isexpressed in plant cells through recombinant DNA technology, asdescribed below. The “antigens” used in practicing the invention may befull-length antigenic proteins from a virus, bacterium, fungus,parasite, protozoan, etc., that causes disease in fish, or alternativelymay constitute an immunogenic portion, fragment or derivative of same. A“derivative” of an amino acid sequence is a sequence related to thereference sequence either on the amino acid sequence level or at the 3Dlevel (i.e. molecules having approximately the same shape andconfiguration as the reference sequence). Derivatives include sequencehomologues, mutants, mimetics, mimotopes, analogues, monomeric forms andfunctional equivalents whether obtained directly from the organism orsynthetically produced, which are capable of inducing an antigenic orimmunogenic response in fish. Particular mention may be made ofderivatives resulting from amino acid substitutions (with natural orsynthetic amino acids), deletions, inversions, insertions, andadditions.

This antigen, whether it is an amino acid sequence or protein, is the“antigen of interest”. The “gene of interest” refers to the nucleotidesequence that encodes for the polypeptide or protein that is the desiredantigen or selection marker. The gene of interest can be optimized forplant transcription and translation by optimizing the codons used forplants (see discussion below).

In general, the methods available for construction of recombinant genesdescribed above, optionally comprising various modifications forimproved expression, can differ in detail. However, conventionallyemployed methods include PCR amplification, or the designing andsynthesis of overlapping, complementary synthetic oligonucleotides,which are annealed and ligated together to yield a gene with convenientrestriction sites for cloning. The methods involved are standard methodsfor a molecular biologist Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Second Edition(1989).

Once the gene is engineered to contain desired features, such as thedesired localization sequences, it is placed into an expression vectorby standard methods. The selection of an appropriate expression vectorwill depend upon the method of introducing the expression vector intohost cells. A typical expression vector contains prokaryotic DNAelements coding for a bacterial origin of replication and an antibioticresistance gene to provide for the growth and selection of theexpression vector in the bacterial host; a cloning site for insertion ofan exogenous DNA sequence, which in this context would code for theantigen of interest; eukaryotic DNA elements that control initiation oftranscription of the exogenous gene, such as a promoter; and DNAelements that control the processing of transcripts, such astranscription termination/polyadenylation sequences. It also can containsuch sequences as are needed for the eventual integration of the vectorinto the plant chromosome.

In a preferred embodiment, the expression vector also contains a geneencoding a selection marker that is functionally linked to a promoterthat controls transcription initiation. By “functionally linked” it isunderstood that the gene of interest (in this case the gene encoding aselection marker) is down-stream of the promoter in the correctorientation and in the correct frame alignment such that transcriptionof mRNA and translation of the mRNA occurs correctly to produce thedesired polypeptide or protein. For a general description of plantexpression vectors and reporter genes, see Gruber et al. (1993) “Vectorsfor Plant Transformation” in Methods of Plant Molecular Biology andBiotechnology CRC Press. p 89-119. In one embodiment, the selective geneis a glufosinate-resistance encoding DNA and in another embodiment canbe the phosphinothricin acetyl transferase (“pat”) or maize optimizedpat gene under the control of the CaMV 35S promoter. The gene confersresistance to bialaphos (Gordon-Kamm (1990) The Plant Cell 2: 603;Uchimiya et al. (1993) Bio/Technology 11: 835; and Anzai et al. (1989)Mol. Gen. Gen. 219: 492).

By “promoter” is meant minimal sequence sufficient to directtranscription. Also included in the invention are those promoterelements which are sufficient to render promoter-dependent geneexpression controllable for cell-type specific, tissue-specific, orinducible by external signals or agents; such elements may be located inthe 5′ or 3′ regions of the gene. Although the endogenous promoter of astructural gene of interest may be utilized for transcriptionalregulation of the gene, the promoter is often a foreign regulatorysequence. Promoter elements employed to control expression of antigenicproteins and the selection gene, respectively, can be anyplant-compatible promoter. Those can be plant gene promoters, such as,for example, the ubiquitin promoter (European patent application no. 0342 926); the promoter for the small subunit ofribulose-1,5-bis-phosphate carboxylase (ssRUBISCO) (Coruzzi, et al.,EMBO J., 3:1671, 1984; Broglie, et al., Science, 224:838, 1984); orpromoters from the tumor-inducing plasmids from Agrobacteriumtumefaciens, such as the nopaline synthase and octopine synthasepromoters (carried on tumor-inducing plasmids of Agrobacteriumtumefaciens and have plant activity); or viral promoters such as thecauliflower mosaic virus (CaMV) 19S and 35S promoters of CaMV (Brisson,et al., Nature, 310:511, 1984; Odell, et al., Nature, 313:810, 1985),the figwort mosaic virus 35S promoter (Gowda, et al., J. Cell Biochem.,13D: 301, 1989) or the coat protein promoter of TMV (Takamatsu, et al.,EMBO J. 6:307, 1987. See also Kay et al. (1987) “Duplication of CaMV 35Spromoter sequences creates a strong enhancer for plant genes” Science236:199-1302 and European Patent Application EP-A-342 926.Alternatively, plant promoters such as the mannopine synthase promoter(Velten, et al., EMBO J., 3:2723, 1984); heat shock promoters, e.g.,soybean hspl7.5-E or hspl 7.3-B (Gurley, et al., Mol. Cell. Biol.,6:559, 1986; Severin, et al., Plant Mol. Biol., 15:827, 1990); orethanol-inducible promoters (Caddick et al., Nature Biotech., 16:177,1998) may be used. See International Patent Application No. WO 91/19806for a review of illustrative plant promoters suitably employed in thepresent invention. In one embodiment of the present invention, the aminoacid-encoding DNA is under the transcriptional control of PGNpr6promoter (WO 01/94394). This is a ubiquitin-like promoter.

In a preferred embodiment, a tissue specific promoter is provided todirect transcription of the DNA preferentially to the seed. One suchpromoter is the globulin promoter. This is the promoter of the maizeglobulin-1 gene, described by Belanger, F. C. and Kriz, A. L. (1991)“Molecular basis for allelic polymorphism of the maize globulin-1 gene”Genetics 129: 863-972. It also can be found as accession number L22344in the Genbank database. Another example is the phaseolin promoter. See,Bustos et al. (1989) “Regulation of B-glucuronidase expression intransgenic tobacco plants by an A/T-rich cis-acting sequence foundupstream of a french bean B-phaseolin gene”, The Plant Cell (1):839-853.

The expression vector can optionally also contain a signal sequencelocated between the promoter and the gene of interest. A signal sequenceis a nucleotide sequence, and possibly the corresponding amino acidsequence, which is used by a cell to direct the protein or polypeptideof interest to be translated and placed in a particular place within oroutside the eukaryotic cell. One example of a plant signal sequence isthe barley α-amylase secretion signal (Rogers, (1985) J. Biol Chem 260,3731-3738). Many signal sequences are known in the art. See, for exampleBecker et al. (1992), Plant Mol. Biol. 20:49; Close, P. S., (1993)Master's Thesis, Iowa State University; Knox, C. (1987), et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17; Lerner et al., (1989) Plant Physiol.91:124-129; Fontes et al. (1991), Plant Cell 3:483-496; Matsuoka et al.(1991), Proc. Natl. Acad. Sci. 88:834; Gould et al. (1989), J. Cell.Biol. 108:1657; Creissen et al. (1991), Plant J. 2:129; Kalderon, et al.(1984) “A short amino acid sequence able to specify nuclear location”Cell 39:499-509; and Steifel, et al. (1990) “Expression of a maize cellwall hydroxyproline-rich glycoprotein gene in early leaf and rootvascular differentiation” Plant Cell 2:785-793.

In one embodiment, the plant selection marker and the gene of interestcan be both functionally linked to the same promoter. In anotherembodiment, the plant selection marker and the gene of interest can befunctionally linked to different promoters. In yet a third and fourthembodiments, the expression vector can contain two or more genes ofinterest that can be linked to the same promoter or different promoters.

Obviously, many variations on the promoters, selectable markers, signalsequences and other components of the construct are available to oneskilled in the art.

In accordance with the present invention, a transgenic plant is producedthat contains a DNA molecule, comprised of elements as described above,integrated into its genome so that the plant can express the gene ofinterest and thus produce the antigen of interest. The transgenic plantmay suitably be a species that is conventionally cultivated for animalfeed, such as corn (Zea mays), canola (Brassica napus, Brassica rapassp.), alfalfa (Medicago sativa), rice (Oyza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower(Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max),potato (Solanum tuberosum), tomatoes (Lycopersicon esculentum), and peas(Lathyrus spp.). Alternatively, the transgenic plant may be a speciesthat is not conventionally eaten, such as tobacco (Nicotiana tabacum),cotton (Gossypium hirsutum), tea (Camellia sinensis), flax, (Linum),sisal (Agave spp., Furcraea spp.), pines, firs and cedars. In order tocreate such a transgenic plant, the expression vectors containing thegene can be introduced into protoplasts, into intact tissues, such asimmature embryos and meristems, into callus cultures, or into isolatedcells. Preferably, expression vectors are introduced into intacttissues. General methods of culturing plant tissues are provided, forexample, by Miki et al. (1993) “Procedures for Introducing Foreign DNAinto Plants” in Methods in Plant Molecular Biology and Biotechnology,Glick et al (eds) CRC Press pp. 67-68 and by Phillips et al. (1988)“Cell/Tissue Culture and In Vitro Manipulation” in Corn and CornImprovement 3d Edit. Sprague et al (eds) American Soc. of Agronomy pp.345-387. The selectable marker incorporated in the DNA molecule allowsfor selection of transformants.

Methods for introducing expression vectors into plant tissue availableto one skilled in the art are varied and will depend on the plantselected. Procedures for transforming a wide variety of plant speciesare well known and described throughout the literature. See, forexample, Miki et al, supra; Klein et al. (1992) Bio/Technology 10:26;and Weisinger et al. (1988) Ann. Rev. Genet. 22: 421-477. For example,the DNA construct may be introduced into the genomic DNA of the plantcell using techniques such as microprojectile-mediated delivery (Kleinet al. (1987) Nature 327: 70-73); electroporation (Fromm et al. (1985)Proc. Natl. Acad. Sci. 82: 5824); polyethylene glycol (PEG)precipitation (Paszkowski et al. (1984) Embo. J. 3: 2717-272); directgene transfer (WO 85/01856 and EP-A-275 069); in vitro protoplasttransformation (U.S. Pat. No. 4,684,611) and microinjection of plantcell protoplasts or embryogenic callus (Crossway, (1985) Mol. Gen.Genetics 202:179-185). Co-cultivation of plant tissue with Agrobacteriumtumefaciens is another option, where the DNA constructs are placed intoa binary vector system (Ishida et al. (1996) “High efficiencytransformation of maize (Zea mays L.) mediated by Agrobacteriumtumefaciens” Nature Biotechnology 14:745-750). The virulence functionsof the Agrobacterium tumefaciens host will direct the insertion of theconstruct into the plant cell DNA when the cell is infected by thebacteria. See, for example Horsch et al. (1984) Science 233: 496-498,and Fraley et al. (1983) Proc. Natl. Acad. Sci. 80: 4803.

Standard methods for transformation of canola are described by Moloneyet al. (1989) “High Efficiency Transformation of Brassica napus UsingAgrobacterium Vectors” Plant Cell Reports 8:238-242. Corn transformationis described by Fromm et al. (1990) Bio/Technology 8:833 and Gordon-Kammet al, supra. Agrobacterium is primarily used in dicots, but certainmonocots such as maize can be transformed by Agrobacterium. See forexample, U.S. Pat. No. 5,550,318. Rice transformation is described byHiei et al. (1994) “Efficient transformation of rice (Oyza sativs L.)mediated by Agrobacterium and sequence analysis of the boundaries of theT-DNA” The Plant Journal 6(2): 271-282, Christou et al. (1992) Trends inBiotechnology 10:239 and Lee et al. (1991) Proc. Nat. Acad. Sci. USA88:6389. Wheat can be transformed by techniques similar to those usedfor transforming corn or rice. Sorghum transformation is described byCasas et al. (1997) “Transgenic sorghum plants obtained aftermicroprojectile bombardment of immature inflorescences” In vitrocellular and developmental biology, Plant. 33:92-100 and by Wan et al.(1994) Plant Physiology. 104:37. Soybean transformation is described ina number of publications, including U.S. Pat. No. 5,015,580.

In one preferred method, the Agrobacterium transformation methods ofIshida supra and also described in U.S. Pat. No. 5,591,616, aregenerally followed, with modifications that the inventors have foundimprove the number of transformants obtained. The Ishida method uses theA188 variety of maize that produces Type I callus in culture. In onepreferred embodiment the Hi II maize line is used which initiates TypeII embryogenic callus in culture. While Ishida recommends selection onphosphinothricin when using the bar or pat gene for selection, anotherpreferred embodiment provides for use of bialaphos instead. In general,as set forth in the '616 patent, and as outlined in more detail below,dedifferentiation is obtained by culturing an explant of the plant on adedifferentiation-inducing medium for not less than seven days, and thetissue during or after dedifferentiation is contacted with Agrobacteriumhaving the gene of interest. The cultured tissue can be callus, anadventitious embryo-like tissue and suspension cells, for example. Inthis preferred embodiment, the suspension of Agrobacterium has a cellpopulation of 10⁶ to 10¹¹ cells/ml and are contacted for three to tenminutes with the tissue, or continuously cultured with Agrobacterium fornot less than seven days. The Agrobacterium can contain plasmid pTOK162,with the gene of interest between border sequences of the T region ofthe plasmid, or the gene of interest may be present in anotherplasmid-containing Agrobacterium. The virulence region may originatefrom the virulence region of a Ti plasmid or Ri plasmid. The bacterialstrain used in the Ishida protocol is LBA4404 with the 40 kb superbinary plasmid containing three vir loci from the hypervirulent A281strain. The plasmid has resistance to tetracycline. The cloning vectorcointegrates with the super binary plasmid. Since the cloning vector hasan E. coli specific replication origin, but not an Agrobacteriumreplication origin, it cannot survive in Agrobacterium withoutcointegrating with the super binary plasmid. Since the LBA4404 strain isnot highly virulent, and has limited application without the superbinary plasmid, the inventors have found in yet another embodiment thatthe EHA101 strain is preferred. It is a disarmed helper strain derivedfrom the hypervirulent A281 strain. The cointegrated superbinary/cloning vector from the LBA4404 parent is isolated andelectroporated into EHA 101, selecting for spectinomycin resistance. Theplasmid is isolated to assure that the EHA101 contains the plasmid.EHA101 contains a disarmed pTi that carries resistance to kanamycin.Hood E E, Helmer G L, Fraley R T, Chilton M D (1986) “The hypervirulenceof Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542outside of T-DNA” J Bacteriol 168: 1291-1301.

Further, the Ishida protocol as described provides for growing freshculture of the Agrobacterium on plates, scraping the bacteria from theplates, and resuspending in the co-culture medium as stated in the '616patent for incubation with the maize embryos. This medium includes 4.3 gMS salts, 0.5 mg nicotinic acid, 0.5 mg pyridoxine hydrochloride, 1.0 mlthiamine hydrochloride, casamino acids, 1.5 mg 2,4-D, 68.5 g sucrose and36 g glucose, all at a pH of 5.8. In a further preferred method, thebacteria are grown overnight in a 1 ml culture, then a fresh 10 mlculture re-inoculated the next day when transformation is to occur. Thebacteria grow into log phase, and are harvested at a density of no morethan OD600=0.5, preferably between 0.2 and 0.5. The bacteria are thencentrifuged to remove the media and resuspended in the co-culturemedium. Since Hi II is used, medium preferred for Hi II is used. Thismedium is described in considerable detail by Armstrong, C. I. and GreenC. E. (1985) “Establishment and maintenance of friable, embryogenicmaize callus and involvement of L-proline” Planta 154:207-214. Theresuspension medium is the same as that described above. All further HiII media are as described in Armstrong et al. The result isredifferentiation of the plant cells and regeneration into a plant.Redifferentiation is sometimes referred to as dedifferentiation, but theformer term more accurately describes the process where the cell beginswith a form and identity, is placed on a medium in which it loses thatidentity, and becomes “reprogrammed” to have a new identity. Thus thescutellum cells become embryogenic callus.

It is preferred to select the highest level of expression of the aminoacid sequence, and it is thus useful to ascertain expression levels intransformed plant cells, transgenic plants and tissue specificexpression. One such method is to measure the expression of the antigenof interest as a percentage of total soluble protein. One standard assayis the Bradford assay which is well known to those skilled in the art(Bradford, M. (1976) Anal. Biochem. 72:248). The biochemical activity ofthe recombinant amino acid sequence should also be measured and comparedwith a wild-type standard.

The levels of expression of the gene of interest can be enhanced by thestable maintenance of the gene of interest on a chromosome of thetransgenic plant. Use of linked genes, with herbicide resistance inphysical proximity to the gene of interest, would allow for maintainingselective pressure on the transgenic plant population and for thoseplants where the genes of interest are not lost.

With transgenic plants according to the present invention, the aminoacid sequence can be produced in commercial quantities. Thus, theselection and propagation techniques described above yield a pluralityof transgenic plants that are harvested in a conventional manner. Theplant seed expressing the recombinant amino acid sequence can be used ina commercial process, or the amino acid sequence can be extracted. Whenusing the seed itself, it can, for example, be made into flour and thenapplied in the commercial process. Extraction from biomass can beaccomplished by known methods. Downstream processing for any productionsystem refers to all unit operations after product synthesis, in thiscase protein production in transgenic seed (Kusnadi et al. (1997)Biotechnology and bioengineering. 56:473-484). Seed is processed eitheras whole seed ground into flour, or fractionated, and the germ separatedfrom the hulls and endosperm. If germ is used, it is usually defattedusing a hexane extraction and the remaining crushed germ ground into ameal or flour. In some cases the germ is used directly or the amino acidsequence can be extracted (See, e.g. WO 98/39461). Extraction isgenerally made into aqueous buffers at specific pH to enhancerecombinant amino acid sequence extraction and minimize native seedprotein extraction. Subsequent amino acid sequence concentration orpurification can follow.

In a further embodiment, plant breeding can be used to introduce thegene into other plants once transformation has occurred. This can beaccomplished by any means known in the art for breeding plants such as,for example, cross pollination of the transgenic plants that aredescribed above with another plant, and selection for plants fromsubsequent generations which express the amino acid sequence. The plantbreeding methods used herein are well known to one skilled in the art.For a discussion of plant breeding techniques, see Poehlman (1987)Breeding Field Crops, AVI Publication Co., Westport Conn. Many cropplants useful in this method are bred through techniques that takeadvantage of the plant's method of pollination. A plant isself-pollinating if pollen from one flower is transferred to the same oranother flower of the same plant. A plant is cross-pollinated if thepollen comes from a flower on a different plant. For example, inBrassica, the plant is normally self sterile and can only becross-pollinated unless, through discovery of a mutant or throughgenetic intervention, self compatibility is obtained. Inself-pollinating species, such as rice, oats, wheat, barley, peas,beans, soybeans, tobacco and cotton, the male and female plants areanatomically juxtaposed. During natural pollination, the malereproductive organs of a given flower pollinate the female reproductiveorgans of the same flower. Maize plants (Zea mays L.) can be bred byboth self-pollination and cross-pollination techniques. Maize has maleflowers, located on the tassel, and female flowers, located on the ear,on the same plant. It can self or cross pollinate.

Pollination can be by any means, including but not limited to hand, windor insect pollination, or mechanical contact between the male fertileand male sterile plant. For production of hybrid seeds on a commercialscale in most plant species pollination by wind or by insects ispreferred. Stricter control of the pollination process can be achievedby using a variety of methods to make one plant pool male sterile, andthe other the male fertile pollen donor. This can be accomplished byhand detassling, cytoplasmic male sterility, or control of malesterility through a variety of methods well known to the skilledbreeder. Examples of more sophisticated male sterility systems includethose described at Brar et al., U.S. Pat. Nos. 4,654,465 and 4,727,219and Albertsen et al. U.S. Pat. Nos. 5,859,341 and 6,013,859.

Backcrossing methods may be used to introduce the gene into the plants.This technique has been used for decades to introduce traits into aplant. An example of a description of this and other plant breedingmethodologies that are well known can be found in references such asPlant Breeding Methodology edit. Neal Jensen, John Wiley & Sons, Inc.(1988). In a typical backcross protocol, the original variety ofinterest (recurrent parent) is crossed to a second variety (nonrecurrentparent) that carries the single gene of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a plant is obtainedwherein essentially all of the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, in addition to the single transferred gene from the nonrecurrentparent.

The preferred method of administration of plant-derived recombinantamino acid sequence to fish is per oral, optionally by admixture of therecombinant amino acid sequence to a conventional feedstuff. Alternativemethods of administration include immersion, intra-peritoneal injection,and intramuscular injection.

Transgenic plant tissue may be fed to the fish, or mixed with othermaterials and fed to fish, or extracted and administered to the fish.

Oral delivery forms of the vaccine encompass any combination of therecombinant amino acid sequence with one or more excipients andoptionally with one or more nutrients. Excipients as used herein caninclude silica, binding agents, emulsions, tensio-active substances,fatty acids, fats, oils etc. and any other additives necessary forpreparing the composition.

Typical fish feedstuffs can comprise various nutrient sources, such as ametabolizable energy source (carbohydrate), a protein source, a fatsource, and optionally fibers, vitamins and minerals. The exactcomposition of the feedstuff depends on the type of fish concerned, andin particular whether or not the fish are carnivorous. On a commercialscale feedstuffs may conveniently be provided in the form of pressed orextruded feed pellets. Plant-derived recombinant amino acid sequence maybe incorporated into the feed by substitution for a more usual proteinsource (such as fish meal, blood meal, maize gluten, soya meal etc.).Alternatively, the plant-derived recombinant amino acid sequence may beadhered to the surface of a pre-formed fish feedstuff.

The plant-derived recombinant amino acid sequence may be enteric-coatedfor oral delivery. The enteric coating protects the vaccine fromproteases and from the relatively low pH levels of the stomach. Thisallows the vaccine to reach the hindgut associated with lymphoid tissue,which maximizes the effectiveness of the vaccine for protecting fish.The enteric coating typically comprises a polymer coating that isunaffected by acidic pH, but which is dissolved upon passing to thehigher pH environments of the intestine.

In a preferred embodiment the plant-derived recombinant amino acidsequence is administered to fish in the form of transgenic plantmaterial, such as plant seeds, leaves, fruits, stems, tubers, etc.,preferably where the transgenic plant material is not admixed to anyother feedstuffs. In another embodiment the plant-derived recombinantamino acid sequence is physically (reversibly) mixed with pre-formedfish feed immediately prior to feeding the fish.

In order to avoid unnecessary extraction procedures, it is preferred todeliver the plant-derived recombinant amino acid sequence in anon-purified (crude) form to the fish. This means that edible parts ofthe source plant are not specially treated or processed in order toextract or concentrate the recombinant amino acid sequence.

The effective dosage of vaccine may vary depending on the size andspecies of the subject, and according to the mode of administration. Theoptimal dosage can be determined through trial and error by aveterinarian or aquaculture specialist. Vaccines may comprise betweenabout 1 and 1000 μg, preferably between about 10 and 200 μg, morepreferably between about 50 and 100 μg of recombinant amino acidsequence in a single dosage.

The vaccine of the invention may be administered to fish forprophylactic or therapeutic purposes. The vaccine is capable of inducinglong term protection against the target infectious disease. “Long term”protection in the case of fish means a protective immune response forlonger than 7 days, more preferably longer than 20 days, and mostpreferably longer than 70 days post vaccination.

Example 1 Transformation of Avidin into Plants and Detection ofExpression Levels Construction of Plasmids for Avidin Expression inPlants

Construction of plasmids for avidin transformation into corn isdescribed in U.S. Pat. No. 5,767,379, incorporated herein by reference.The chicken egg white avidin cDNA was reported by Gope M L. (1987), etal., Nuc. Acids Res. 15: 3595-3606. The amino acid sequence is reversetranslated into nucleic acid sequence utilizing a preferred maize codonusage table (GCG, assembled by Mike Cherry, Stanford University). Fromthis computer-generated synthetic sequence, overlapping, complementaryoligonucleotides with compatible restriction site termini are designed,then annealed and ligated to yield the maize optimized gene. Thesequence used is set forth in the '379 patent, incorporated byreference. The barley alpha amylase signal sequence ((Rogers, (1985) J.Biol Chem 260, 3731-3738) is also synthesized (using overlapping,complementary nucleotides) with maize-preferred codons. Compatiblerestriction sites between these two gene fragments are ligated, with thebarley alpha amylase signal sequence at the 5′ end of the avidin geneand in proper frame alignment so that the correct codon usage occursduring translation to yield the desired antigen. The resultant barleyalpha amylase signal sequence/avidin segment is cloned, (See FIG. 1 (SEQID NO: 1)) as a BamHI/EcoRI fragment, into the vector pGEM3Zf+, aproduct of Promega Corporation (Madison, Wis.), to generate plasmidpPHI5142. A BamHI/HpaI fragment containing the barley alpha amylasesignal sequence/avidin region is isolated and cloned into a plasmidderived from pBlueScript SK+ (Stratagene, La Jolla, Calif.), as abackbone. In this plasmid, the signal sequence/avidin gene fragment isinserted, in the correct orientation, between the maize ubiquitin 5′region, which includes the maize ubiquitin promoter (UBI1ZM), the firstexon and first intron, and the potato proteinase inhibitor II (PinII)transcription terminator region (An et al, (January 1989 (Plant Cell 1:115-122). The resultant plasmid is pPHI5168 (FIG. 2). Co-transformedwith the plasmid is a plasmid (pPHI610) containing the bar gene fromStreptomyces hygroscopicus, supra and White J. (1990) Nucleic Acids Res18:1062 linked to the double 35S promoter (e.g. Friz, S. E. J. Cell Sci98:545-550), the intron from the maize alcohol dehydrogenase gene(Callis J., et al. Genes and Development 1:1183-1200) and the PinIIterminator (An G., et al. (1989) Plant Cell 1:115-122). These constructsand the process used are fully described in the '379 patent, supra. Notethat in the experiment described in the '379 patent, the bar gene isused, where in the other experiments described herein the maizeoptimized pat gene is used. FIG. 3 sets forth this sequence (SEQ ID NO:2).

Transformation and Tissue Culture to Produce Avidin-Expressing Plants.

An established callus line derived from a single immature embryo of the“Hi II” maize plants (Armstrong C L, Green C E, Phillips R L (1991)Maize Gen. Coop. Newsletter, 65:92-93) is transformed using particlebombardment-mediated transformation with a helium-powered particleacceleration device, PDS 1000 (Bio-Rad, Hercules, Calif.). Hi II is acorn plant line used in research frequently because of its ease intransformation. Tissue showing a friable type-II embryogenic morphologyis sieved through 710 m mesh prior to co-transformation with equimolaramounts of the avidin gene (pPHI5168) and the bar selectable marker gene(PHP610), according to the procedures of Tomes et al. (Tomes D T, Ross MC, Songstad D D (1995) Plant Cell Tissue and Organ Culture: FundamentalMethods. Springer-Verlag, Berlin, Heidelberg. pp. 197-213).Transformants expressing the bar gene are selected in the presence ofbialaphos (3 mg 1-1), according to the protocol of Register et al.(Register J. C.-III et al. (1994), Plant Mol. Biol. 25:951-961).Co-transformants that also express the avidin gene are identified byELISA screening of the selected colonies. Multiple plants (T₀generation) are regenerated from avidin-expressing colonies, transferredto the greenhouse and assayed for avidin expression in leaf tissue.) T₁seed is obtained by outcrossing, with the T₀ plants as the female parentand a non-transformed inbred line (PHN46; see U.S. Pat. No. 5,567,861)as the male parent.

ELISA to Detect Avidin in Corn.

The following procedures are used to detect expression of avidin inseeds. Seeds are powdered and extracted in 10 mM PBS pH 7.0 containing0.05% Tween-20 (PBST). Total protein was quantified using the Bradfordmicrotiter assay Bradford (Bradford, M. M. 1976. A rapid and sensitivemethod for the quantitation of microgram quantities of protein utilizingthe principal of protein-dye binding. Anal. Biochem. 72:248-254). ELISAsare typical sandwich style in which the microtiter plates are coatedwith rabbit anti-avidin antibody, the avidin protein is capturedovernight at 4° C., and the plate is reacted with goat anti-avidinantibody (Vector Labs, Burlingame, Calif.) followed by anti-goatalkaline phosphatase conjugate (Jackson Immunoresearch, West Grove,Pa.). The alkaline phosphatase is detected with para-nitrophenylphosphate and read at 405 nm on a SpectroMax plate reader (MolecularDevices, Sunnyvale, Calif.).

Example 2 Transformation of LtB into Plants and Detection of Expression

LtB sequences and introduction into plants is described at U.S. Pat. No.6,194,560, which sequences and methods were used in this experiment, andwhich is incorporated herein by reference. The vector used here differsin certain aspects from that described in the '560 patent. It isPGN7101, shown in FIG. 4. The LtB gene of an E. coli strain of humanorigin (Leong et al. (1985) Nucleotide sequence comparison betweenheat-labile toxin B-subunit cistrons from Escherichia coli of human andporcine origin Infect Immun. April; 48(1):73-7) is synthesized tooptimize codon usage for maize, see FIG. 5A (SEQ ID NO: 3).Oligonucleotides spanning the gene are annealed and ligated, and theproducts are amplified using the polymerase chain reaction (PCR). Anoligonucleotide sequence encoding the barley α-amylase secretion signal(BAASS) is added at the N-terminus of LtB using PCR and this completeBAASS:LtB sequence fragment is inserted into a vector backbone resultingin the plasmid PGN5431. The BAASS:LtB sequence is shown in FIG. 5B (SEQID NO: 4). The BAASS:LtB sequence is removed from PGN5431 using therestriction enzymes NcoI and HpaI and ligated into the correspondingrestriction sites in the vector PGN2774 resulting the intermediatevector PGN7020. In this intermediate vector, the BAASS:LtB is placed 3′to a maize constitutive promoter and untranslated leader sequence fromthe ubiquitin regulatory system, designated PGNpr1 (wild type maizepolyubiquitin-1), and 5′ to the potato proteinase inhibitor IItranscription terminator (PinII). The BAASS:LtB expression cassette(promoter, leader, BAASS:LtB and Pin II sequences) is removed fromPGN7020 using the restriction enzymes NheI and NotI and ligated into thecorresponding sites in the plant transformation vector PGN3770. Thefinal BAASS:LtB transformation vector, designated PGN7101, contains theright and left border sequences of Agrobacterium tumefaciens Ti plasmidorigin, and the pat gene of Streptomyces viridichromogenes, conferringresistance to glufosinate ammonium.

Example 3 Transformation of IPNV into Plants and Detection of Expression

Infectious pancreatic necrosis virus (IPNV) infects mollusks,crustaceans and many types of fish, especially salmonids. IPNV infectioncan have devastating effects on salmonid production due to fishmortality at the fry or smolt stage and decreased growth in survivingpopulations. There have been many attempts to produce an effectivevaccine against this virus. So far protection has been seen only with aninjected inactivated virus, however this vaccine has proven to beexpensive and impractical. The major structural and immunogenic proteinsof the virus, VP2 and VP3, are expressed in maize using the methodsdescribed, supra.

Nucleotide sequences for VP2 and VP3 are initially obtained from theplasmids pUK-NVP2 and pUK-NVP3 respectively. The sequences for theproteins in these two plasmids are from a Norwegian IPNV strain closelyrelated to the N1 strain. Some nucleotide modification is carried out onthe 5′ and 3′ ends of the gene sequences to optimize codon usage formaize.

An oligonucleotide sequence encoding 5′ VP2 sequences, that are missingfrom the VP2 gene in pUK-NVP2, along with nucleotide changes for codonoptimization, is annealed at the 5′ end of the VP2 sequence frompUK-NVP2 using polymerase chain reaction (PCR). An oligonucleotidesequence encoding nucleotide changes for codon optimization at the 3′end of VP2 along with sequences from the potato proteinase inhibitor IItranscription terminator (PinII) (An et al., Plant Cell (1989)1:115-122) is added at the 3′ end of the VP2 sequence from pUK-NVP2using PCR. These two PCR fragments along with an internal VP2 fragment,isolated using the restriction enzymes SacII and BbsI, from pUK-NVP2 areligated together to give the plasmid PGNK5676 containing the completeVP2 nucleotide sequence (SEQ ID NO: 5) shown in FIG. 6. Anoligonucleotide sequence encoding the barley α-amylase secretion signal(BAASS) is added at the N-terminus of the restored VP2 gene using PCR.The fragment generated from PCR is put into a vector backbone resultingin the plasmid PGNK5443 containing the BAASS:VP2 nucleotide sequence(SEQ ID NO: 6) shown in FIG. 7.

Oligonucleotides encoding nucleotide changes for codon optimization formaize are annealed to both the 5′ and 3′ ends of the VP3 sequences, frompUK-NVP3, using PCR. The PCR fragment is put into a vector backbone togive the plasmid PGNK5581 containing the partially optimized VP3sequence (SEQ ID NO: 7) shown in FIG. 8. An oligonucleotide sequenceencoding BAASS is added to the N-terminus of VP3 using PCR. The PCRfragment is put into a vector backbone to give the plasmid PGNK5330containing the BAASS:VP3 sequence (SEQ ID NO: 8) shown in FIG. 9.

Two separate plant transformation vectors are constructed, eachcontaining both of the genes for VP2 and VP3. The first constructcontains the sequences for BAASS:VP2 and BAASS:VP3, each in a separateexpression cassette containing a maize seed preferred promoter,designated PGNpr2, and the PinII terminator. The BAASS:VP2 sequences arecut from PGNK5443 with the restriction enzymes NcoI and PacI. Thisfragment along with the PGNpr2 fragment cut with the restriction enzymesHindIII and NcoI are ligated into the HindIII and PacI restriction sitesof the PGN9004 plant transformation vector which contains the PinIIterminator, the right and left border sequences of Agrobacteriumtumefaciens Ti plasmid origin, and the pat gene of Streptomycesviridichromogenes, conferring resistance to glufosinate ammonium. Thisplasmid is designated PGNK5461. In a similar process the BAASS:VP3sequences are cut from PGNK5330 using NcoI and PacI. This fragment alongwith the HindIII/NcoI PGNpr2 fragment is ligated into the HindIII andPacI sites of PGN9004 resulting in the plasmid PGNK5335. The BAASS:VP2expression cassette, containing the PGNpr2 promoter, BAASS, VP2 and thePinII terminator, is cut from PGNK5461 using the restriction enzymesAscI and PacI. The BAASS:VP3 expression cassette, containing the PGNpr2promoter, BAASS, VP3 and the PinII terminator, is cut from PGNK5335using the restriction enzymes HindIII and MluI. These two fragments areligated into the HindIII and PacI restriction sites of PGN9004 resultingin the final plant transformation vector containing both the BAASS:VP2and BAASS:VP3 expression cassettes. This construct, designated PGN9084(FIG. 10), is designed such that the proteins are sent to the cell walland accumulate primarily in the seed. The plants are then transformedaccording to the modified Ishia protocol, set forth supra. Plantsresulting from the transformation of PGN9084 are designated NVA.

The second plant transformation vector also contains both VP2 and VP3 inseparate expression cassettes under the control of the PGNpr2 promoterand the PinII terminator, however the barley α-amylase secretion signal(BAASS) is not present. A 5′ portion of the VP2 sequence up to andincluding the BstBI restriction site is cut from PGNK5573 using therestriction enzymes BbsI and BstBI. This fragment and the HindIII/NcoIPGNpr2 fragment are ligated into the HindIII and BstBI sites in PGNK5461resulting in the plasmid PGNK5676 containing the VP2 expressioncassette. The VP3 sequence is cut from PGNK5581 using the restrictionenzymes NcoI and PacI. This fragment and the HindIII/NcoI PGNpr2fragment are ligated into the HindIII and PacI sites of PGN9004resulting in the plasmid PGNK5681 containing the VP3 expressioncassette. The VP2 expression cassette is cut from PGNK5676 using therestriction enzymes AscI and PacI. The VP3 expression cassette is cutfrom PGNK5681 using the enzymes HindIII and MluI. These two fragmentsare ligated into the HindIII and PacI restriction sites of PGN9004resulting in the final plant transformation vector containing both theVP2 and VP3 expression cassettes. This construct, designated PGN9111(FIG. 11), is designed such that the proteins are sent to the cytoplasmand accumulate primarily in the seed. Plants resulting from thetransformation of PGN9111 are designated NVB.

Western blot analysis using polyclonal anti-IPNV whole virus antibodiesshows expression of the proteins VP2 and VP3 in both NVA and NVB seed.The VP2 and VP3 proteins expressed in NVA seed run slightly larger thanthe corresponding native proteins found in the IPNV whole virus standardon a Western blot (FIG. 12). Lane 1 shows protein markers, lanes 2-4 apurified prep of IPNV whole virus, lane 5 control maize seed extract,negative control, lanes 6-11 extracts from various NVA seed and lane 12an unpurified prep of IPNV whole virus. (Note, the purification processof the whole virus generates the smearing pattern in the top of thosewells)

Since the VP2 and VP3 proteins are targeted by the BAASS to the cellwall in the NVA seed, it is expected that the proteins will beglycosylated. Not wishing to be bound by theory, it is possible thatthis increase in size of both proteins suggests glycosylation of theproteins in the plant. Both VP2 and VP3 expressed in NVB seed run at theexpected sizes compared to the IPNV whole virus standard on a Westernblot (FIG. 13). Lane 1 shows protein markers, lane 2 extract from NVAseed, lanes 3-9 extracts from various NVB seed, and lanes 10-12increasing amounts of unpurified IPNV whole virus.

Since these proteins are expressed in the cytoplasm of the plant cell,no modification of the proteins is expected.

Expression levels of VP2 and VP3 in the NVA seed are measured by meansof a Western blot. The intensity of the VP2 and VP3 protein bands fromthe seed extracts are measured using spot densitometry and are thencompared to the bands of known amounts of whole virus. Using this methodthe expression of VP2 in NVA T2 seed is calculated to be 0.1% TSP (totalsoluble protein) and the expression of VP3 in NVA T2 seed is calculatedto be 0.3% TSP. Expression levels of VP2 in the NVB seed are measured byELISA. The ELISA is a typical sandwich style in which the microtiterplate is coated with sheep anti-IPNV whole virus antiserum, the IPNVprotein in the plant extract is captured overnight at 4° C., and theplate is reacted with AS1 mouse monoclonal anti-VP2 antibody followed byalkaline phosphatase conjugated sheep anti-mouse IgG. The alkalinephosphatase is detected with para-nitrophenyl phosphate, disodium (pNpp)and read at 405 nm on an absorbance microplate reader. Using his methodthe expression of VP2 in NVB T1 seed is measured to be 0.17% TSP in thehighest single seeds.

Example 4 Feeding Studies with Avidin and LtB

To evaluate this new technology in fish, this experiment is designed todetermine if oral administration of diets containing corn-expressedrecombinant marker proteins induces a humoral immune response insalmonids. Atlantic salmon are fed, in an amount of approximately 2% ofbody weight per day, diets containing two doses of unpurified cornexpressing LtB (5% or 10% of food) or chicken egg white avidin (10% or20% of food) for 5 days, 12 days with normal food and 5 days with thetreated diet. Groups of fish are also intraperitoneally injected withpurified LtB and avidin protein as positive controls.

Fish growth, persistence of recombinant proteins in feces and humoralimmune response are examined. Fish antibody response is compared for thedifferent doses of LtB, which has been shown previously to be capable ofproducing a strong antibody response in mice, and avidin, which has beenshown previously to be a weaker antigen in mice.

The Atlantic salmon weigh about 20 grams each. There are a total ofeleven treatment groups with several negative and positive controlgroups (Table 1). A and B positive control groups, each consisting often fish, are given an intraperitoneal injection with oil-adjuvantedpreparations with group A receiving a single injection of 4 μg LtBprotein per fish and group B receiving a single injection of 20 μgavidin protein per fish which are both recombinant proteins purifiedfrom a corn expression system. Nine groups each contain 35 fish, withgroup C to G being negative controls. Group C receives commercial fishpellets. Group D receives pellets which include 5% non-transgenic corngerm. Group E receives pellets with 10% non-transgenic corn germ. GroupF receives pellets made with fish meal having 10% non-transgenic cornflour and Group G receives pellets with 20% non-transgenic corn flour.In the experimental groups, Group H receives fish pellets with 5% LtBtransgenic corn germ, and group I receives pellets with 10% LtB corngerm. Group J receives pellets with 10% transgenic avidin-containingflour, and group K receives pellets with 20% avidin flour.

TABLE 1 Number Weight (g) Total protein Feed (g) for Group Vaccine ofFish corn required amount (mg) 10 days A Injected Lt-B positive 10 00.040* 40 control B Injected Avidin 10 0 0.20* 40 positive control CNegative Control 1 35 0 0 140 (normal food) D Negative Control 25% 35 00 140 normal corn germ meal E Negative Control 2 35 0 0 140 10% normalcorn germ meal F Negative Control 4 35 14 0 140 10% normal corn flour GNegative Control 5 35 28 0 140 20% normal corn flour H 5% recombinantLtB 35 7 2.1 140 corn germ meal I 10% recombinant LtB 35 14 4.2 140 corngerm meal J 10% avidin corn flour 35 14 20.61 140 K 20% avidin cornflour 35 28 41.22 140 *Each fish is given an intraperitoneal (ip)injection of 0.1 ml PBS containing 4 μg purified recombinant Lt-B (groupA), or 20 μg avidin (group B) in an oil adjuvant.

Fish are maintained at 10° C. At two, four, seven, fourteen and twentyone days post-feeding five fish are sacrificed in nine diet groups C-K,to measure the persistence of the recombinant proteins in the fecesusing an ELISA. At eight weeks, ten fish in all eleven groups aresacrificed weighed and specific antibody in the serum is measured byELISA

ELISA to Detect Marker Proteins in Feces:

ELISA is the typical sandwich style in which the microtiter plates arecoated overnight at 4° C. with rabbit anti-avidin or anti-LtB antibody.Fish fecal samples, diluted in PBST, are added to wells and the plateincubated overnight at 4° C. to allow capture of the avidin or LtBprotein. The plate is reacted with goat anti-avidin antibody (VectorLabs, Burlingame, Calif.) or mouse biotinylated LtB antibody followed byanti-goat alkaline phosphatase conjugate (Jackson Immunoresearch, WestGrove, Pa.) or ExtraAvidin-alkaline phosphatase (Sigma-Aldrich CanadaLtd., Oakdale, ON). The alkaline phosphatase is detected withpara-nitrophenyl phosphate (Pierce, distributor MJS Biolynx Inc.,Brockville, ON, Canada) and read at 405 nm on a plate reader (BioTekInstruments Inc., Vermont).

ELISA to Detect Specific Anti-Avidin and Anti-LtB Antibodies in FishSerum:

A sandwich ELISA is used to detect specific antibodies in fish serum.Plate wells are coated with purified LtB or purified corn-expressedavidin (Sigma) overnight at 4° C., two-fold dilutions of fish serum inPBST-1% BSA are added and the plates incubated overnight at 17° C. toallow fish antibody capture. Primary antibody, monoclonal anti-Atlanticsalmon Ig (Cedarlane Laboratories Ltd., ON, Canada), followed bysecondary alkaline-phosphatase labeled anti-mouse Ig antibody(Cedarlane), both diluted in PBST-1% BSA, are added to the plates. Afterthe addition of para-nitrophenyl phosphate substrate (Pierce),absorbance is read at 405 nm using a microtiter plate reader (BioTekInstruments). Antibody titer is calculated as the endpoint dilution.

The addition of ground corn expressing the two marker proteins to thediet does not affect fish growth as shown in FIG. 14. The two markerproteins are detectable for an extended time period in the feces, atleast 21 days after cessation of feeding the treated diet as shown inTable 2.

TABLE 2 Day Post-Feeding Group 2 4 7 14 21  5% LtB 5/5 4/4 5/5 4/5 NS10% LtB 5/5 4/4 3/4 3/4 2/4 10% avidin 5/5 5/5 4/5 5/5 4/4 20% avidin3/3 4/4 5/5 4/4 4/4

Oral administration of the marker proteins induces a humoral immuneresponse. At 8 weeks post-vaccination, only fish in the negative controlgroups do not have a detectable specific serum antibody response. Theantibody response of fish fed unpurified corn-expressed marker proteinsis as strong as those of fish injected with pure proteins in oiladjuvant as shown in FIG. 15.

Example 5 Feeding Studies with IPNV

The methods described in feeding the corn containing infectiouspancreatic necrosis virus VP2 and VP3 proteins will be completed. Thepresence of the viral proteins in the feces and organs of the animal isexpected, as well as antibody responses After fish are challenged withvirulent virus it is expected that oral administration of thecorn-expressed IPNV proteins will result in protection.

Fish will be divided into seven groups and tagged for identification.Positive control group A will consist of fish given an injection with acommercial vaccine that induces protection against IPNV and negativecontrol group B will be fed commercial food pellets. The remaininggroups will be fed food containing corn germ with and without expressedIPNV proteins for 5 days, 12 days with normal food and 5 days with foodcontaining corn germ as outlined in Table 3. The percent incorporationrate of corn germ into food (g corn per g food) will be 10% and 20%.

TABLE 3 Group Number of fish Treatment A 55 ip injected commercialvaccine B 55 normal food C 85 non-transgenic corn germ mixed into food20% incorporation D 85 NVA corn germ 10% incorporation E 85 NVA corngerm 20% incorporation F 85 NVB corn germ 10% incorporation G 85 NVBcorn germ 20% incorporation

At 4 weeks post-vaccination, all fish will acclimated over a few days tosalt water and then maintained in flowing salt water at ambienttemperature (9-12° C.). At 5 weeks post-transfer to salt water, fishwill be exposed to a cohabitation IPNV challenge. Naive fish will beinjected with live IPNV and added to tanks containing the vaccinatedfish. Daily mortality will be monitored for five weeks. On a weeklybasis from 1 to 5 weeks post-vaccination and at the time of challenge, 5fish per groups C to G will be sacrificed and sampled for feces andorgans to examine IPNV protein persistence and distribution. Fish killedat the time of challenge, including 5 fish in groups A and B, will alsobe bled and the serum tested for antibodies by ELISA.

1. A plant comprising a recombinant nucleotide sequence encoding anamino acid sequence integrated into the plant genome, the sequenceencoding an amino acid which, when administered to a fish, results in aprotective response in said fish wherein the nucleotide sequence is asequence selected from the group consisting of SEQ ID NO: 6, 7, and 8.2. A plant seed comprising a recombinant nucleotide sequence of claim 1integrated into the plant seed genome, the sequence encoding an aminoacid sequence which, when administered to a fish, results in aprotective response in said fish.
 3. A plant cell comprising arecombinant nucleotide sequence of claim 1 integrated into the plantcell genome, the sequence encoding an amino acid sequence which, whenadministered to a fish, results in a protective response in said fish.4. The plant of claim 1 wherein the amino acid sequence is an antigen ofan organism that causes disease or pathology in a fish.
 5. A compositionfor administration to a fish, comprising plant material comprising arecombinant nucleotide sequence integrated into the genome of the plantmaterial, the sequence encoding an amino acid sequence which, whenadministered to a fish, results in a protective response in said fishwherein the nucleotide sequence is a sequence selected from the groupconsisting of SEQ ID NO: 6, 7, and
 8. 6. The composition of claim 5wherein the plant material comprises seed tissue comprising therecombinant nucleotide sequence.
 7. The composition of claim 5 whereinthe plant material is combined with at least one nutrient or excipient.8. The composition of claim 5 wherein the amino acid sequence is anantigen of an organism that causes disease or pathology in fish.
 9. Theplant of claim 1, wherein the plant is a monocotyledonous plant.
 10. Theplant of claim 1, wherein the plant is corn.
 11. The plant of claim 1,wherein the plant is a dicotyledonous plant.
 12. The plant of claim 1,wherein the amino acid is expressed in the plant at levels of at leastabout 0.01% total soluble protein.
 13. The plant of claim 1, wherein theamino acid is expressed in the plant at levels of at least about 0.1%total soluble protein.
 14. The plant cell of claim 3, wherein the cellis a monocotyledonous plant cell.
 15. The plant cell of claim 3, whereinthe cell is a corn cell.
 16. The plant cell of claim 3, wherein the cellis a dicotyledonous plant cell.
 17. The plant cell of claim 3 furthercomprising a second nucleotide sequence which causes the amino acid tobe secreted to the cell wall.
 18. A method of producing a compositionfor administration to a fish, comprising transforming a plant with anucleotide sequence encoding an amino acid sequence which, whenadministered to a fish, results in a protective response in said fish.19. The method of claim 18 wherein the amino acid sequence an antigen ofan organism that causes disease or pathology in fish
 20. The method ofclaim 18 wherein the amino acid sequence is extracted from the plant.21. The method of claim 18 wherein the transgenic plant is crossed withat least one plant to produce progeny comprising the amino acidsequence.
 22. The method of claim 18 wherein the nucleotide sequence isselected from the group consisting of SEQ ID NO: 5, 6, 7, and
 8. 23. Amethod of producing a composition for administration to a fishcomprising providing biomass from a plurality of plants, of which atleast certain plants comprise a heterologous nucleotide sequenceencoding an amino acid sequence which, when administered to a fish,results in a protective response in said fish, wherein the nucleotidesequence is operably linked to a promoter to effect expression of theprotein by the certain plants.
 24. The method of claim 23 wherein theamino acid sequence is an antigen of an organism that produces diseaseor pathology in fish.
 25. The method of claim 23 wherein the nucleotidesequence is selected from the group consisting of SEQ ID NO: 5, 6, 7,and
 8. 26. A method of inducing an immune response in fish comprisingfeeding fish a plant or plant material from a plant comprising anucleotide sequence encoding an amino acid sequence which, whenadministered to a fish, results in a immune response in said fish. 27.The method of claim 26 wherein the amino acid sequence is from anantigen of an organism causing disease or pathology in fish.
 28. Themethod of claim 27 wherein the nucleotide sequence is selected from thegroup consisting of SEQ ID NO: 5, 6, 7, and
 8. 29. A method of treatinga fish infected with an organism that causes disease or pathology infish, comprising administering to the fish a composition comprisingplant material comprising a recombinant nucleotide sequence integratedinto the plant genome, the sequence encoding an amino acid sequencewhich is an antigen of the organism causing disease or pathology in fishand, which, when administered to a fish, results in a protectiveresponse in said fish.
 30. The method of claim 20 wherein saidcomposition contains a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 5, 6, 7, and
 8. 31. A method of protecting afish from disease or pathology comprising orally administering to thefish a composition comprising plant material comprising a recombinantnucleotide sequence integrated into the genome of the plant material,the sequence encoding an amino acid sequence which protects the fishfrom disease or pathology.
 32. The method of claim 31 wherein the aminoacid is expressed in the plant material at levels of at least about0.01% total soluble protein.
 33. The method of claim 31 wherein theamino acid is expressed in the plant material at levels of at leastabout 0.1% total soluble protein.
 34. The method of claim 31 wherein thenucleotide sequence encodes an amino acid protecting the fish frominfectious pancreatic necrosis virus.
 35. The method of claim 31,wherein the nucleotide sequence encodes VP2 or VP3.
 36. The method ofclaim 35, wherein the VP2 or VP3 is expressed in the plant at levels ofat least about 0.1% total soluble protein.
 37. A composition foradministration to a fish, comprising plant material comprising arecombinant nucleotide sequence integrated into the genome of the plantmaterial, the sequence encoding an amino acid which, when administeredto a fish, results in a protective response in said fish, wherein theamino acid is expressed at levels of at least about 0.01% total solubleprotein.
 38. The composition of claim 37 wherein the amino acid isexpressed at levels of at least about 0.1% total soluble protein.